Gene associated with non-biological stress resistance, and transformed plant

ABSTRACT

The present invention relates to a composition for enhancing non-biological stress resistance in plants and a composition for accelerating germination. A nucleotide sequence of the present invention is involved in the resistance against the drying stresses in plants, and a transformed plant in which the nucleotide sequence is overexpressed has prominent resistance against various kinds of non-biological stress, including drought stress. In addition, the nucleotide sequence of the present invention is involved in ABA hormone sensitivity in plants, and germination is greatly improved in a plant in which the nucleotide sequence expression is suppressed. Therefore, the composition of the present invention can be useful as new functional crops regardless of the climate of the cultivation area, or as seeds for long-term storage with an increased storage period.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a gene implicated in abiotic stresstolerance and growth promotion and a method for improving abiotic stresstolerance and promoting growing of transformed plants with the same.

2. Description of the Related Art

Due to their sessile nature, higher plants are constantly faced withvarious adverse environmental factors, including drought, high salt,heavy metals, cold, heat shock, and ozone, during their whole life span.These abiotic stresses are a limiting factor for the growth anddevelopment of crop plants. Water deficiency causes dramatic reductionof crop production globally, and the decreasing availability of freshwater may pose a future threat to humans and higher plants. Plants havediverse defense strategies to enhance their tolerance to transient andlong-term water shortages by triggering signaling network pathways andinducing stress-responsive genes. The cellular and genetic defensemechanisms in response to water stress have been widely documented(Shinozaki and Yamaguchi-Shinozaki, 2007). However, for stress toleranceor sensitivity, our knowledge concerning the biological functions ofstress-related genes in higher plants is still rudimentary. Therefore,it is important to study the functions of stress responsive genes toincrease the productivity and distribution of crop plants.

Ubiquitin is a protein consisting of 76 amino acids and it has beenfound in almost all tissues of eukaryotic organisms. Ubiquitin has acharacteristic that is covalently bound to various substrate proteins byE1-E2-E3 consecutive actions of ubiquitin-activating enzymes (E1s),ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Thesubstrate proteins to be attached with ubiquitin are very diverse,affecting almost all physiological activities. In addition, many studieshave been found that the many diseases are associated with thesemechanisms. A function of ubiquitin is firstly known to promotedegradation of protein by attaching with other proteins. However, otherfunctions of ubiquitin have been recently revealed one after another.

Ubiquitin is attached to substrate by consecutive actions of three typesof proteins, i.e., E1, E2 and E3. The glycine residue at the C-terminaldomain of ubiquitin binds to NH₃ at R— group of lysine residues on thesubstrate protein, thereby forming a covalent bond with the substrate.In general, proteins attached with ubiquitin are degraded by proteasome.Polyubiquitin as a chain of several ubiquitin molecules has to beattached to the substrate for degradation by proteasome. Until now, ithas been known that proteasome-dependent degradation of the substrateoccurs only when polyubiquitin consisting of at least four ubiquitins isattached to the substrate; however, it would be controversial sincethese results were obtained from in vitro experiments.Polyubiquitination leading to the proteasome-dependent degradation isthe linkage form in which the 48^(th) lysine residue of one ubiquitin islinked to another ubiquitin.

There are 2 types of E1 enzymes in organism. There are various types ofE2s. In general, E2s catalyse the transfer of ubiquitin from E1 to E3 orsubstrate. E3s which are also known as E3 ligases catalyse the finalstep of the ubiquitination cascade. E3s determine specificity of thesubstrate to be ubiquitinated. In other words, the substrate beingcapable of interaction with certain E3s is specifically determined. E3enzymes may be classified into two major types according to domains. E3enzymes possess one of two domains: the homologous to the E6-AP carboxylterminus (HECT) domain and the really interesting new gene (RING)domain. E3 enzymes having RING domain serves to position E2 andsubstrate in close proximity each other. In other words, where E2 andthe substrate bind to E3, distance between ubiquitin of E2 and thesubstrate is formed to close sufficiently such that ubiquitin of E2 ischemically passed to the substrate. In contrast, E3 enzymes having HECTdomain receive ubiquitin from E2, and then transfer it to the substrate.The At5g01520 gene codes for the protein having E3 ubiquitin ligaseenzymatic activity. The ubiquitination has been known to serve diversefunctions as one of the mechanism that all higher organisms as well asplants have. However, the genes involved in abiotic stresses have beenunknown. The present inventors have isolated the At5g01520 genes inwhich the expression is induced by abiotic stresses and ABA hormone inArabidopsis thaliana. Then, they have prepared At5g01520s-overexpresorsand knock-out mutants and analyzed their physiological phenotypes.

Throughout this application, various publications and patents arereferred and citations are provided in parentheses. The disclosures ofthese publications and patents in their entities are hereby incorporatedby references into this application in order to fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive studies to improve a toleranceto abiotic stresses of a plant. As results, they have discovered that anucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 wasinvolved in the above-mentioned characteristic of the plant. Inaddition, where the gene expression was inhibited, transgenic plantshaving the improved tolerance to abiotic stresses may be obtained.

Accordingly, it is an object of this invention to provide a compositionfor improving the tolerance of a plant to an abiotic stress, and a plantcell or a plant exhibiting improved tolerance to an abiotic stress,transformed with the composition.

It is another object of this invention to provide a composition forpromoting germination of a plant.

Other objects and advantages of the present invention will becomeapparent from the following detailed description together with theappended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-d represent results of analyzing the AtAIRP2 gene expression byRT-PCR after treatments of various abiotic stresses and ABA hormone.After treatments of ABA hormone (FIG. 1a ), drought stress (FIG. 1b ),low-temperature stress (FIG. 1c ) and salt stress (FIG. 1d ), each RNAwas extracted to analyze the gene expression pattern. RD29A was used asa representative control gene in treatments of drought, salt andlow-temperature, and RAB18 was used as a representative control gene intreatment of ABA.

FIG. 2 represents results of analyzing the promoter activity of theAtAIRP2 gene by GUS assay. When 100 μM ABA (3 hours) or drought (2hours) condition was treated, GUS signals were markedly induced. Itcould be determined that promoter activity of the AtAIRP2 gene wasincreased.

FIG. 3 represents results of analyzing the enzymatic activity of theAtAIRP2 protein. Maltose-binding protein (MBP) was bound to the AtAIRP2proteins. Then, MBP-AtAIRP2 was incubated with HIS-UBA1, HIS-UBC8,ubiquitin and AtAIRP2 at 30° C. for 1 hour to performSelf-Ubiquitination, and performed by Western blot using MBP- andubiquitin-specific antibodies to analyze changes in the protein levels.As a result, it was determined that the molecular weight of the AtAIRP2protein was increased through Western blot using anti-MBP antibody, andthe increase was induced due to ubiquitin. Based on the results, itcould be demonstrated that the AtAIRP2 protein possessed ability forenzymatic activity of E3 ubiquitin ligase that binds ubiquitin protein.

FIGS. 4a-d represent results of measuring the AtAIRP2 gene mutants andtheir tolerance to drought stress. FIG. 4a represents the gene map thatthe T-DNAs were inserted to the exon (AtAIRP2-1) and the intron(AtAIRP2-2) in genomic DNA of the AtAIRP2 gene. FIG. 4b represents thatT-DNA insertions were verified by PCR amplification using the T-DNAborder primer and primers annealing to sites upstream and downstream ofthe T-DNA insertion site with the extracted genomic DNA from theknock-out mutant. FIG. 4c represents that the expression of the gene wasanalyzed by RT-PCR with the extracted RNA from the knock-out mutant.Based on the results, it could be demonstrated that the expression ofthe gene in the AtAIRP2 mutant was inhibited. FIG. 4d represents imagesof comparing the tolerance to drought stress in the AtAIRP2 gene mutantsand the wild type Arabidopsis thaliana. Each of plants was grown for 2weeks, and subjected to drought stress by withholding water for 13 days,respectively. The plants were then re-watered and monitored the numberof the survived plant. As a result, mutants were less tolerant todrought stress than the wild types (FIG. 4d ).

FIGS. 5a-b represent results of the AtAIRP2-overexpressing transgenicplants. Arabidopsis thaliana was transformed by 355:AtAIRP2-GFPrecombinant vector and it was verified whether to overexpress the geneusing anti-GFP antibody (FIG. 5a ). As a result of Western blot, itcould be demonstrated that the AtAIRP2-GFP protein was well-expressed.FIG. 5b represents images of comparing the tolerance to drought stressin the AtAIRP2-overexpressing transgenic and the wild type Arabidopsisthaliana. Each of plants was grown for 2 weeks, and subjected to droughtstress by withholding water for 14 days, respectively. The plants werethen re-watered and monitored the number of the survived plant. As aresult, the AtAIRP2-overexpressing transgenic plants were more tolerantto drought stress than the wild types.

FIGS. 6a-b represent results of analyzing germination rates according toABA hormone in the AtAIRP2 knock-out mutant and theAtAIRP2-overexpressing transgenic plant. FIG. 6a represents images ofthe wild type, the AtAIRP2-1 mutant and the AtAIRP2-2 mutant which weregrown on medium supplemented with different concentrations (0.1 and 0.5μM) of ABA hormone for 7 days. It could be understood that thegermination rates of mutants which were grown on medium supplementedwith ABA hormone were higher than that of the wild types. FIG. 6brepresents images of the wild type, the AtAIRP2-2 mutant and theAtAIRP2-sGFP overexpressing transgenic plant which were grown on mediumsupplemented with different concentrations (0.2 and 0.4 μM) of ABAhormone for 7 days. It could be understood that the germination rates ofthe AtAIRP2 overexpressing transgenic plants which were grown on mediumsupplemented with ABA hormone were significantly inhibited. In addition,it could be demonstrated that the AtAIRP2-2 mutant showed the tolerantunder the same condition. Therefore, it could be understood that thegermination rates in the AtAIRP2-overexpressing transgenic plants weredecreased by ABA hormone and the germination rate in mutant wasincreased by ABA hormone.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of this invention, there is provided a composition forimproving the tolerance of a plant to an abiotic stress, comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.

The present inventors have made intensive studies to improve a toleranceto abiotic stresses of a plant. As results, they have discovered that anucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 wasinvolved in the above-mentioned characteristic of the plant. Inaddition, where the gene expression was inhibited, transgenic plantshaving the improved tolerance to abiotic stresses may be obtained.

According to a preferred embodiment, the present nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:2 comprises the nucleotidesequence as set forth in SEQ ID NO:1. According to the presentinvention, the nucleotide sequence as set forth in SEQ ID NO:1 is thenucleotide sequence of the At5g01520 gene in Arabidopsis thaliana, andthe gene is named as the AtAIRP2 (Arabidopsis thaliana ABA InsensitiveRing Protein 2). The gene encodes RING protein having E3 ubiquitinligase enzymatic activity. The present inventors have found thatexpressions of the gene were increased by various abiotic stresses andABA hormone.

It would be obvious to the skilled artisan that the nucleotide sequencesused in this invention are not limited to those listed in the appendedSequence Listings.

For nucleotides, the variations may be purely genetic, i.e., ones thatdo not result in changes in the protein product. This includes nucleicacids that contain functionally equivalent codons, or codons that encodethe same amino acid, such as six codons for arginine or serine, orcodons that encode biologically equivalent amino acids.

Considering biologically equivalent variations described hereinabove,the nucleic acid molecule of this invention may encompass sequenceshaving substantial identity to them. Sequences having the substantialidentity show at least 80%, more preferably at least 90%, mostpreferably at least 95% similarity to the nucleic add molecule of thisinvention, as measured using one of the sequence comparison algorithms.Methods of alignment of sequences for comparison are well-known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J.Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins andSharp, CABIOS 5:151-3(1989) Corpet et al., Nuc. Acids Res.16:10881-90(1988) Huang et al., Comp. Appl. BioSci. 8:155-65(1992) andPearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI Basic LocalAlignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol.215:403-10(1990)] is available from several sources, including theNational Center for Biological Information (NBCI, Bethesda, Md.) and onthe Internet, for use in connection with the sequence analysis programsblastp, blasm, blastx, tblastn and tblastx. It can be accessed athttp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determinesequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to a preferred embodiment, the present abiotic stress isselected from the group consisting of a drought stress, alow-temperature stress and a salt stress.

According to the present invention, the present inventors havedemonstrated that expressions of the At5g01520 gene were increased whenplants were subjected to drought stress, low-temperature stress or saltstress. In addition, they have demonstrated that the tolerances to thesestresses were improved when the gene was over-expressed in plants.

More preferably, the present abiotic stress is drought stress.

In another aspect of this invention, there is provided a composition forimproving a tolerance of a plant to an abiotic stress, comprising arecombinant plant expression vector which comprises: (a) the nucleotidesequence as disclosed in the present invention; (b) a promoter which isoperatively linked to the nucleotide sequence of (a) and generates a RNAmolecule in plant cells; and (c) a poly A signal sequence inducingpolyadenylation at the 3′-end of the RNA molecule.

The term “operatively linked” as used herein refers to functionallinkage between a nucleic acid expression control sequence (such as apromoter, signal sequence, or array of transcription factor bindingsites) and a second nucleotide sequence, wherein the expression controlsequence affects transcription and/or translation of the nucleic acidcorresponding to the second sequence.

The vector system of this invention may be constructed in accordancewith conventional techniques described in Sambrook et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001), teachings of which are incorporated herein by reference.

The suitable promoter in the present invention includes any one commonlyused in the art, for example SP6 promoter, T7 promoter, T3 promoter, PMpromoter, maize-ubiquitin promoter, Cauliflower mosaic virus (CaMV)-35Spromoter, Nopalin synthase (nos) promoter, Figwort mosaic virus 35Spromoter, Sugarcane bacilliform virus promoter, commelina yellow mottlevirus promoter, photo-inducible promoter of small subunit ofRibulose-1,5-bis-phosphate carboxylase (ssRUBISCO), cytosolictriosphosphate isomerase (TPI) promoter in rice, adeninephosphoribosyltransferase (APRT) or octopine synthase promoter inArabidopsis. Preferably, the promoter used in this invention is CaMV35S.

According to a preferred embodiment, the poly A signal sequence inducingpolyadenylation at the 3′-end includes that from the nopaline synthasegene of Agrobacterium tumefaciens (NOS 3′ end) (Bevan et al., NucleicAcids Research, 11(2):369-385(1983)), that from the octopine synthasegene of Agrobacterium tumefaciens, the 3′-end of the protease inhibitorI or II genes from potato or tomato, the CaMV 35S terminator, and OCS(octopine synthase) terminator. Most preferably, the poly A signalsequence inducing polyadenylation at the 3′-end in this invention is OCS(octopine synthase) terminator.

Optionally, the present vector for plants may further carry a reportermolecule (e.g., genes for luciferase and β-glucuronidase). In addition,the vector may contain antibiotic resistant genes as selective markers(e.g., neomycin phosphotransferase gene (nptII) and hygromycinphosphotransferase gene (hpt)).

According to a preferred embodiment, the plant expression vector of thisinvention is Agrobacterium binary vectors.

The term “binary vector” as used herein, refers to a cloning vectorcontaining two separate vector systems harboring one plasmid responsiblefor migration consisting of left border (LB) and right border (RB), andanother plasmid for target gene-transferring. Any Agrobacterium suitablefor expressing the nucleotide of this invention may be used, and mostpreferably, the transformation is carried out using Agrobacteriumtumefaciens GV3101.

Introduction of the recombinant vector of this invention intoAgrobacterium can be carried out by a large number of methods known toone skilled in the art. For example, particle bombardment,electroporation, transfection, lithium acetate method and heat shockmethod may be used. Preferably, the electroporation is used.

In still another aspect of this invention, there is provided a plantcell exhibiting improved tolerance to an abiotic stress, transformedwith the composition of this invention.

In further aspect of this invention, there is provided a plantexhibiting improved tolerance to an abiotic stress, transformed with thecomposition of this invention.

To introduce a foreign nucleotide sequence into plant cells or plantsmay be performed by the methods (Methods of Enzymology, Vol. 153 (1987))known to those skilled in the art. The plant may be transformed usingthe foreign nucleotide inserted into a carrier (e.g., vectors such asplasmid or virus) or Agrobacterium tumefaciens as a mediator (Chilton etal., Cell, 11:263:271 (1977)) and by directly inserting the foreignnucleotide into plant cells (Lorz et al., Mol. Genet., 199: 178-182(1985); the disclosure is herein incorporated by reference). Forexample, electroporation, microparticle bombardment, polyethyleneglycol-mediated uptake may be used in the vector containing no T-DNAregion.

Generally, Agrobacterium-mediated transformation is the most preferable(U.S. Pat. Nos. 5,004,863, 5,349,124 and 5,416,011), and the skilledartisan can incubate or culture the transformed cells or seeds to matureplants in appropriate conditions.

The term “plant(s)” as used herein, is understood by a meaning includinga plant cell, a plant tissue and a plant seed as well as a mature plant.

The plants applicable of the present invention include, but not limitedto, food crops such as rice plant, wheat, barley, corn, bean, potato,Indian bean, oat and Indian millet; vegetable crops such as Arabidopsissp., Chinese cabbage, radish, red pepper, strawberry, tomato,watermelon, cucumber, cabbage, melon, pumpkin, welsh onion, onion andcarrot; crops for special use such as ginseng, tobacco plant, cottonplant, sesame, sugar cane, sugar beet, Perilla sp., peanut and rape;fruit trees such as apple tree, pear tree, jujube tree, peach tree, kiwifruit tree, grape tree, citrus fruit tree, persimmon tree, plum tree,apricot tree and banana tree; flowering crops such as rose, gladiolus,gerbera, carnation, chrysanthemum, lily and tulip; and fodder crops suchas ryegrass, red clover, orchardgrass, alfalfa, tallfescue and perennialryograss.

In still further aspect of this invention, there is provided acomposition for promoting germination of a plant comprising a nucleicacid molecule, wherein nucleic acid molecule inhibits an expression of anucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.

According to a preferred embodiment, the present nucleotide sequencecomprises the nucleotide sequence as set forth in SEQ ID NO:1.

According to the present invention, the nucleotide sequence as set forthin SEQ ID NO:1 is the nucleotide sequence of the At5g01520 gene.According to the present invention, it was determined that Whereexpressions of the present nucleotide sequence were inhibited, thesensitivity to ABA hormone suppressing immature-germination wasdecreased such that the germination rates were increased. Therefore, thepresent nucleic acid molecule enables to store seeds for a long timewith excellent germination rates.

According to a preferred embodiment, the nucleic acid molecule is T-DNA,siRNA, shRNA, miRNA, ribozyme, PNA (peptide nucleic acids) or antisenseoligonucleotide. More preferably, the present nucleic acid molecule isT-DNA.

The term “siRNA” used herein refers to a short double strand RNA thatenables to mediate RNA interference via cleavage of mRNA. The siRNA ofthe present invention may consist of a sense RNA strand having asequence corresponding to a target gene and an antisense RNA strandhaving a sequence complementary to the target gene. The siRNA to inhibitexpression of a target gene provides effective gene knock-down method orgene therapy method.

The siRNA of this invention is not restricted to a RNA duplex of whichtwo strands are completely paired and may comprise non-paired portionsuch as mismatched portion with non-complementary bases and bulge withno opposite bases. The overall length of the siRNA is 10-100nucleotides, preferably 15-80 nucleotides, and more preferably, 20-70nucleotides. The siRNA may comprise either blunt or cohesive end so longas it enables to inhibit the target gene expression via RNAi effect. Thecohesive end may be prepared in 3′-end overhanging structure or 5′-endoverhanging structure. The base number protruded is not particularlylimited, for example 1-8 bases, preferably 2-6 bases. In addition, siRNAmay comprise low molecular weight RNA (for example, tRNA, rRNA, naturalRNA molecule such as viral RNA or artificial RNA molecule) in theprotruded portion of one end to the extent that it enables to maintainan effect on the inhibition of target gene expression. The terminalstructure of siRNA is not demanded as cut structure at both ends, andone end portion of double strand RNA may be stem-and-loop structurelinked by a linker RNA. The length of linker is not restricted where ithas no influence on the pair formation of the stem portion.

The term “shRNA” used herein refers to a single strand nucleotideconsisting of 50-70 bases, and forms stem-loop structure in vivo. LongRNA of 19-29 nucleotides is complementarily base-paired at bothdirections of loop consisting of 5-10 nucleotides, forming adouble-stranded stem.

The term “miRNA (microRNA)” functions to regulate gene expression andmeans a single strand RNA molecule composed of 20-50 nucleotides infull-length, preferably 20-45 nucleotides, more preferably 20-40nucleotides, much more preferably 20-30 nucleotides and most preferably,21-23 nucleotides. The miRNA is an oligonucleotide which is notexpressed intracellularly, and forms a short stem-loop structure. ThemiRNA has a whole or partial complementarity to one or two or more mRNAs(messenger RNAs), and the target gene expression is suppressed by thecomplementary binding of miRNA to the mRNA thereof.

The term used herein “ribozyme” refers to a RNA molecule having anactivity of an enzyme in itself which recognizes and restricts a basesequence of a specific RNA. The ribozyme consists of a binding portioncapable of specifically binding a base sequence complementary to atransfer RNA strand and an enzymatic portion to cut target RNA.

The term “PNA (peptide nucleic acid)” used herein refers to a moleculehaving the characteristics of both nucleic acid and protein, which iscapable of complementarily binding to DNA or RNA. PNA was first reportedin 1999 as similar DNA in which nucleobases are linked via a peptidebond (Nielsen P E, Egholm M, Berg R H, Buchardt O, “Sequence-selectiverecognition of DNA by strand displacement with a thymine-substitutedpolyamide”, Science 1991, Vol. 254: pp 1497-1500). PNA is absent in thenatural world and artificially synthesized through a chemical method.PNA is reacted with a natural nucleic acid having a complementary basesequence through hybridization response, forming double strand. In thedouble strand with the same length, PNA/DNA and PNA/RNA double strandare more stable than DNA/DNA and DNA/RNA double strand, respectively.The form of repeating N-(2-aminoethyl)-glycine units linked by amidebonds is commonly used as a basic peptide backbone. In this context, thebackbone of peptide nucleic acid is electrically neutral in comparisonto that of natural nucleic acids having negative charge. Four bases ofnucleic acid present in PNA are almost the same to those of naturalnucleic acid in the respect of spatial size and distance betweennucleobases. PNA has not only a chemical stability compared with naturalnucleic acid, but also a biological stability due to no degradation by anuclease or protease.

The term “antisense oligonucleotide” used herein is intended to refer tonucleic acids, preferably, DNA, RNA or its derivatives, that arecomplementary to the base sequences of a target mRNA, characterized inthat they bind to the target mRNA and interfere its translation toprotein. The antisense oligonucleotide of the present invention refersto DNA or RNA sequences which are complementary to a target mRNA,characterized in that they bind to the target mRNA and interfere itstranslation to protein, translocation into cytoplasm, maturation oressential activities to other biological functions. The length ofantisense nucleic acids is in a range of 6-100 nucleotides andpreferably 10-40 nucleotides.

The antisense oligonucleotides may be modified at above one or morepositions of base, sugar or backbone to enhance their functions [DeMesmaeker, et al., Curr Opin Struct Biol., 5(3): 343-55 (1995)]. Theoligonucleotide backbone may be modified with phosphothioate,phosphotriester, methyl phosphonate, single chain alkyl, cycloalkyl,single chain heteroatomic, heterocyclic bond between sugars, and so on.In addition, the antisense nucleic acids may include one or moresubstituted sugar moieties. The antisense oligonucleotides may include amodified base. The modified base includes hypoxanthine, 6-methyladenine,5-me pyrimidine (particularly, 5-methylcytosine),5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC,2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil,5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N6(6-aminohexyl)adenine, 2,6-diaminopurine, and so forth.

The term “T-DNA” used herein refers to a DNA fragment as a transfer DNAin Ti (tumor-inducing) plasmid of Agrobacterium sp., which istransferred into a nucleus of a host plant cell. A 25 bp repeat sequenceis present in both termini of T-DNA, and DNA transfer proceeds at thedirection from a left border to a right border. A bacterial T-DNA withabout 20,000 in length destroys a target gene by insertion, resulting ininsertional muatagenesis. In addition to mutation, inserted T-DNAsequence may label a target gene. According to the present invention,the present inventors have used seeds of Arabidopsis thaliana forsuppressing the expression of the At5g01520 gene by means of Ti-plasmidtransformation.

In still further aspect of this invention, there is provided acomposition for promoting germination of a plant, comprising arecombinant plant expression vector which comprises: (a) the nucleotidesequence as disclosed in the present invention; (b) a promoter which isoperatively linked to the nucleotide sequence of (a) and generates a RNAmolecule in plant cells; and (c) a poly A signal sequence inducingpolyadenylation at the 3′-end of the RNA molecule.

Since the nucleic acid molecule, the plant expression recombinant vectorand the introduction method thereof are mentioned above, they areomitted herein to avoid excessive overlaps.

The features and advantages of the present invention will be summarizedas follows:

(a) The present invention provides a composition for improving thetolerance of a plant to an abiotic stress and a composition forpromoting germinating of a plant.

(b) The present nucleotide sequence is involved in tolerance to droughtstress of plants. Therefore, the overexpressing transgenic plants haveexcellent tolerances to various abiotic stresses including droughtstress, whereby they may be useful as novel functional crops which areaffected by climates and environments of the cultivated areas.

(c) In addition, the present nucleotide sequence is involved insensitivity to ABA hormone of plants. Therefore, germination abilitiesof the knock-out mutant plants in which the expression is inhibited areremarkably enhanced, whereby they may effectively used for cultivatingthe plants with novel function of storage period increasing, andbiomass.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES Experimental Methods Isolation of the Genes

The present inventors have isolated the AtAIRP2 genes inducible by ABAhormone and abiotic stresses from cDNAs of Arabidopsis thaliana.10-day-old Arabidopsis thaliana seedlings were grinded with liquidnitrogen in mortar. The powders were added with 2 ml of an extractionbuffer (4 M guanidine-HCl 20 mM, 10 mM EDTA, 10 mM EGTA (USB), 0.5%Sarkosyl (SIGMA), pH 9) and β-mercaptoethanol (SIGMA-ALDRICH) per 1 g ofthe powder for extraction. The extraction resultant was transferred tonew conical tube, suspended with an equal volume of PCI(phenol:chloroform:isoamyl alcohol=25:24:1), vortexed for 5 min andcentrifuged at 3,500 rpm for 25 min (Hanil centrifuge, HA-1000-3). Aftercentrifugation, the upper organic solvent phase was removed. The extractwas resuspended with an equal volume of PCI, vortexed and centrifugedtwice. Then, the lower aqueous phase was undergone twice ethanolprecipitation and once LiCl precipitation to isolate RNA. RNA wasquantified. Single-strand cDNA was synthesized by using 2 μg of RNA witholigo dT primer and MMLV reverse transcriptase (Fermentas). PCR wasconducted in the final volume of 50 μL containing 20 ng of cDNA as atemplate, 10 pmole of each of two types of primers, 5 μL of 10× Taqpolymerase buffer (Takara), 8 μL of dNTPs (each of 1.25 mM) and 1 unitof Taq DNA polymerase (Takara). The tube containing the reaction mixturewas placed in Perkin Elmer DNA thermal cycler. The sequences of primersused in this Example are as follows: 5′-ATGCGAAAATCGTTCAAGGA-3′ (AtAIRP2ORF FW: SEQ ID NO: 3) and 5′-TCACCGAGGAAGAGGAGCATAA-3′ (AtAIRP2 ORF RV:SEQ ID NO: 41). The reaction mixture was denatured for 2 min at 94° C.and subjected to 30 cycles of 30 sec at 94° C., 30 sec at 52° C. and 1min at 72° C. After 30 cycles, polymerization was further performed at72° C. for 5 min. Then, the AtAIRP2 gene amplification was verified byusing electrophoresis method. In addition, the DNA was confirmed bysequencing.

Plant Growth Conditions and Sampling

In order to prepare the AtAIRP2-overexpressing transgenic plants,Invitrogen gateway system was used to construct. First, AtAIRP1-sGFP wasintroduced into pENTR SD Topo vector (Invitrogen, USA) and subsequentlyintegrated into pEarlygate 100 vector (Arabidopsis research center, USA)of plants using LR clonase enzyme (Invitrogen).

Seeds of the AtAIRP2 knock-out mutants (seed number: SAIL_686_G08(AtAIRP2-1), Salk_005082 (AtAIRP2-2)) which are T-DNA insertion lineswere purchased from SIGNAL Salk Institute Genomic Analysis Laboratory(http://signal.salk.edu/).

The seeds of the AtAIRP2-overexpressing transgenic plants, knock-outmutants and the wild type Arabidopsis thaliana were soaked in 30% bleachsolution (Yuhanclorox) and 0.025% Triton X-100 for 10 min, and washed 10times with sterilized water. The treated seeds were grown on MS(Murashige and Skoog) medium (Duchefa Biochemie) that contained 3%sucrose, B5 vitamin (12 mg/L) and 0.8% agar (pH 5.7) in a growth chamberfor 2 weeks (under a condition of 16 hrs-light/8 hrs-dark cycle). Wheregreen whole plants of light condition were used as materials, seeds weregrown on soil of Sunshine MIX #5 (Sun GroHorticulture) in a growthchamber for 3 weeks (under a condition of 16 hrs-light/8 hrs-darkcycle).

Treatments of Stresses (Salt, Low-Temperature, Drought and ABA Hormone)

In order to determine expressions of the AtAIRP2 gene to drought stress,the wild type Arabidopsis thaliana seedlings which were grown on mediumfor 2 weeks were exposed in the air, and sampled after 1 hour and 2hours. In order to determine expressions of the AtAIRP2 gene to saltstress, the wild type Arabidopsis thaliana seedlings which were grown onmedium for 2 weeks were treated with 300 mM sodium chloride, and sampledafter 1.5 hour and 3 hours. In order to determine expressions of theAtAIRP2 gene to low-temperature stress, the wild type Arabidopsisthaliana seedlings which were grown on medium for 2 weeks were incubatedat 4° C. for 12 hours and 24 hours, and sampled. In order to determineexpressions of the AtAIRP2 gene to ABA hormone stress, the wild typeArabidopsis thaliana seedlings which were grown on medium for 2 weekswere treated with 100 μM of ABA (SIGMA), and sampled after 1.5 hour and3 hours. The sampled tissues were grinded with liquid nitrogen inmortar. The powders were added with β-mercaptoethanol (SIGMA-ALDRICH)and 2 ml of an extraction buffer (4 M guanidine-HCl 20 mM, 10 mM EDTA,10 mM EGTA (USB), 0.5% Sarkosyl (SIGMA), pH 9) per 1 g of the powder toextract. The extract was transferred to new conical tube, suspended withan equal volume of PCI (phenol:chloroform:isoamyl alcohol=25:24:1),vortexed for 5 min and centrifuged at 3,500 rpm for 25 min (Hanilcentrifuge, HA-1000-3). After centrifugation, the supernatant which isupper organic solvent phase was removed. The extract was resuspendedwith an equal volume of PCI, vortexed and centrifuged. The extract wasperformed twice with the process described above. Then, the loweraqueous phase was performed twice with ethanol precipitation and oncewith LiCl precipitation to isolate RNA.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from leaves of the AtAIRP2-overexpressingtransgenic plants, knock-out mutants and the wild type Arabidopsisthaliana. Single-strand cDNA was synthesized by using 2 μg of RNA witholigo dT primer and MMLV reverse transcriptase (Fermentas). PCR wasconducted in the final volume of 50 μL containing 20 ng of cDNA as atemplate, 10 pmole of each of two types of primers, 5 μL of 10× Taqpolymerase buffer (Intron), 8 μL of dNTPs (each of 1.25 mM) and 1 unitof Taq DNA polymerase (Intron). The tube containing the reaction mixturewas placed in Perkin Elmer DNA thermal cycler. The reaction mixture wasdenatured for 2 min at 94° C. and subjected to 25 cycles of 30 sec at94° C., 30 sec at 52° C. and 1 min at 72° C. After 25 cycles,polymerization was further performed at 72° C. for 5 min. Then, the PCRproducts were stored at −20° C. in a freezer. The sequences of primersused in this Example are shown in Table 1.

TABLE 1 Primers used in RT-PCR Primer sequence AtAIRP2 RT FW5′-GATGGTGGCTACGTTCAGA-3′ (SEQ ID: 5) AtAIRP2 RT RV5′-AAATGTCAATAACCAATGGTTG-3′ (SEQ ID: 6) Rab18 FW5′-GCGTCTTACCAGAACCGTCC-3′ (SEQ ID: 7) Rab18 RV 5′-CCCTTCTTCTCGTGGTGC-3′(SEQ ID: 8) RD29a FW 5′-CAGGTGAATCAGGAGTTGTT-3′ (SEQ ID: 9) RD29a RV5′-CCGGAAATTTATCCTCTTCT-3′ (SEQ ID: 10) UBC10 FW5′-TGGATATGGCGTCGAAGC-3′ (SEQ ID: 11) UBC10 RV5′-GTGGGATTTTCCATTTAGCC-3′ (SEQ ID: 12)Extraction of Genomic DNA of Mutants Inserted with T-DNA and Acquisitionof Homozygous Mutant

The seeds of the wild type Arabidopsis thaliana and knock-out mutantswere grown on soil for 2 weeks and their leave were sampled. The leaveswere grinded with liquid nitrogen in mortar. The powders were added with700 mL of CTAB buffer (2% CTAB, 100 mM Tris pH 8, 20 mM EDTA, 1.4 MNaCl, 2% PVP), mixed and heated at 65° C. for 10 min. The resultantswere added to 200 mL of chloroform, mixed and centrifuged. Aftercentrifugation, the supernatant was removed. The resultant was mixedwith isopropanol to precipitate DNA. The precipitate was washed with 70%ethanol, dried. The obtained genomic DNA was dissolved in water to use.Genotyping PCR was performed using T-DNA border primer (LB_6313R) andprimers annealing to sites upstream and downstream of the T-DNAinsertion site.

TABLE 2 Primers used in Genotyping PCR and RT-PCR Primer SequenceLB_6313R 5′-GAGCTGCTATACACTGATCTGAG-3′ (SEQ ID: 13) AtAIRP2 FW15′-CGTGTGCTCTACGCGAATC-3′ (SEQ ID: 14) AtAIRP2 RV15′-CCCTTAATCAGCAAATATGATATCG-3′ (SEQ ID: 15)

It was revealed that the T-DNA insertions were mapped to the second exonand the first intron in the AtAIRP2 gene knock-out mutant, which wasverified by PCR amplification using the T-DNA border primer and primersannealing to sites upstream and downstream of the T-DNA insertion site(FIG. 4b ). In addition, the suppression of the gene expression wasanalyzed by RT-PCR using AtAIRP2 FW1 and AtAIRP2 RV1 primers with theextracted RNA from the knock-out mutant (FIG. 4c ).

Preparation of Vector Construct of the AtAIRP2 Gene

For construction of a recombinant plasmid for expressing the fusionprotein between the AtAIRP2 and maltose-binding protein (MBP), PCR wascarried out using a primer set designed to contain XbaI and PstIrestriction sites linked to 5′-direction and 3′-direction of the codingregion of the AtAIRP2 gene, respectively. PCR products and pMAL-X vector(New England Biolabs, Beverly, Mass.) were restricted by XbaI and PstIrestriction enzymes and then ligated using T4 DNA ligase (New EnglandBio Lab). The recombinant MBP-AtAIRP2 was expressed in Escherichia colistrain BL21-CodonPlus (DE3) RIL (Stratagene) and purified using amylosecolumn chromatography. The protein was quantified using BSA as astandard protein. In addition, in the present invention, Invitrogengateway system was used to construct for preparing transgenic plants.First, AtAIRP2-sGFP was introduced into pENTR SD Topo vector(Invitrogen, USA) and subsequently integrated into pEarlygate 100 vector(Arabidopsis research center, USA) of plants using LR clonase enzyme(Invitrogen).

AtAIRP2 Transformation with Agrobacterium tumefaciens Strain andPreparation of the AtAIRP2 Transgenic Plants

The prepared constructs were transferred to Agrobacterium tumefaciensstrain GV3101 by electroporation. The presence of the gene was confirmedby PCR. An aerial part of approximately 4-week-old Arabidopsis thaliana(columbia [Col-0]) was soaked on MS medium (Duchefa Biochemie)containing 0.05% Silwet for 1.5 min to transform (clough and Bent, 1998,Plant J 16; 735-743). The seedlings were grown for 3 weeks in a 23°C.-growth chamber to obtain seeds (T1). The transformed seeds (T1) wereselected from medium containing 25 μg/mL of BASTA (Glufosinate ammonium)and 250 μg/mL of carbenicillin. The presence of the transgene wasverified by RT-PCR and western blot. Overexpression of the transgene wasobserved using anti-GFP antibody (clontech).

Analysis on Enzymatic Activity of the AtAIRP2 Protein

For the enzymatic activity analysis of the AtAIRP2 protein, the ORF ofthe AtAIRP2 gene was subcloned into pMAL-X vector in-frame with MBP(maltose-binding protein). 40 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP,2 mM dithiothreitol (DTT), 300 ng/μL ubiquitin (Sigma), 25 μM MG132(A.G. Scientific Inc.), 500 ng UBA1 (ABRC, http://www.arabidopsis.org),500 ng UBC8 (ABRC, http://www.arabidopsis.org) and 500 ng MBP-AtAIRP2were added to each of tubes and incubated at 30° C. After addition ofsample buffer solution, the resultant was heated at 100° C. for 5 min,and performed by Western blot using anti-MBP (New England Bio Labs) andanti-ubiquitin (Santa Cruz) antibodies.

Comparison of Plant Growth

For comparing phenotypes to ABA hormone, seeds obtained from the wildtype, the AtAIRP2 knock-out mutant and the AtAIRP2-overexpressingtransgenic plant were grown on medium supplemented with differentconcentrations (0, 0.2, 0.4 and 0.5 μM) of ABA hormone for 7 days, andtheir germination degrees were then measured. In order to comparephenotypes to salt, seeds obtained from the wild type, the AtAIRP2knock-out mutant and the AtAIRP2-overexpressing transgenic plant weregrown on medium supplemented with different concentrations (0, 0.2, 0.4and 0.5 μM) of sodium chloride for 7 days, and their germination degreeswere then measured.

Measurement of Sensitivity of Adult Plants to Drought Stress

Seeds obtained from the wild type, the AtAIRP2 knock-out mutant and theAtAIRP2-overexpressing transgenic plant were grown on soil for 2 weeks,and subjected to drought stress by withholding water for 13 or 14 days,respectively. The plants were then re-watered and measured the degreesof the tolerance to drought stress.

Histochemical GUS Assay

The wild type Arabidopsis thaliana was grown on medium for 10 days,subjected to 100 mM of ABA hormone and drought stress, and fixed with90% acetone for 5 min. After removal of the acetone, the plant waswashed 3 times with rincing solution containing 50 mM NaPO₄, 1 mMK₃Fe(CN)₆, and 1 mM K₄Fe(CN)₆, and immersed in 2 mM X-Gluc(5-bromo-4-chloro-3-indolyl glucuronide, sigma) and vacuumed for 1 min.After staining at 37° C. in the dark until the color was changed, theplant was incubated in 90% ethanol to remove chlorophyll.

Experimental Results

The AtAIRP2 Gene Expression after Stress Treatments (Salt,Low-Temperature and Drought)

The AtAIRP2 gene expressions in various abiotic stresses were analyzedby RT-PCR. After treatments of low-temperature (12 hours and 24 hours),drought (1 hour and 2 hours), salt (1.5 hour and 3 hours) and ABAhormone (1.5 hour and 3 hours) stresses, RNA from each of samples wasextracted to analyze the gene expression patterns. As a result, it wasdetermined that the gene expression levels of stress treatments wereincreased than those of non-stress treatments, thereby the AtAIRP2 geneexpression is induced by salt, low-temperature, drought and ABA hormonestresses (FIG. 1). In addition, the AtAIRP2 protein expressions invarious abiotic stresses were analyzed by histochemical GUS assay. Aftertreatments of ABA hormone (3 hours) and drought (2 hours) stresses, thedegree of the staining was analyzed. As a result, it was determined thatthe degrees of the staining of stress treatments were increased thanthose of before the stress treatments (FIG. 2).

Analysis on Enzymatic Activity of the AtAIRP2 Protein

Maltose-binding protein (MBP) was bound to the AtAIRP2 proteins. Then,the MBP-AtAIRP2 was incubated with UBA1, UBC8, ubiquitin and AtAIRP2 at30° C. for 1 hour to perform Self-Ubiquitination, and performed byWestern blot using MBP- and ubiquitin-specific antibodies to analyzechanges in the protein levels. As a result, it was determined that themolecular weight of the AtAIRP2 protein was increased through Westernblot using anti-MBP antibody, and the increase was induced due toubiquitin (FIG. 3). Based on the results, it could be demonstrated thatthe AtAIRP2 protein possessed ability for enzymatic activity of ligasethat binds ubiquitin protein.

Acquisition of the AtAIRP2 Mutants

As shown in FIG. 4a , the gene that the T-DNA insertions were mapped tothe second exon and the first intron was used in order to prepare themutants. In order to determine whether to insert and position of T-DNA,genotyping PCR was performed using T-DNA boder primer and primersannealing to sites upstream and downstream of the T-DNA insertion site.As a result, it was determined that T-DNA was inserted in the samedirection as that of the gene (FIG. 4b ). In addition, in order todetermine whether to express mRNA of the full-length AtAIRP2 gene inmutants, the same primers used in genotyping PCR were used. As a result,it was determined that the AtAIRP2 gene was not expressed (FIG. 4c ).

Measurement of Sensitivity of Adult Plants to Drought Stress

In order to measure the tolerance to drought stress in the AtAIRP2knock-out mutant and the wild type Arabidopsis thaliana, each of theplants was grown for 2 weeks, and subjected to drought stress bywithholding water for 13 days, respectively. The plants were thenre-watered and monitored the number of the survived plant. As a result,knock-out mutants survived respectively by 25% and 35% whereas the wildtypes survived by 85%. Therefore, it could be demonstrated that themutants were less tolerant to drought stress than the wild types (FIG.4d ). In order to measure the tolerance to drought stress in theAtAIRP2-overexpressing transgenic plants, homozygous plants of each oftransgenic plants obtained through Basta selection were acquired. Theobtained seeds were grown on medium. The protein expressions intransgenic plants of #10 and #19 were verified by Western Blot usinganti-GFP (FIG. 5a ). In order to compare the tolerance to drought stresson the AtAIRP2-overexpressing transgenic plant and the wild typeArabidopsis thaliana, each of the plants was grown for 2 weeks, andsubjected to drought stress by withholding water for 14 days,respectively. The plants were then re-watered and monitored the numberof the survived plant. As a result, the AtAIRP2-overexpressingtransgenic plant #10 and #19 survived respectively by 87.5% and 84.2%whereas the wild types survived by 10%. Therefore, it could bedemonstrated that the transgenic plants were more tolerant to droughtstress than the wild types (FIG. 5b ).

Comparison of Plant Germination Rate

As a result of comparison on germination rate in the wild type and theAtAIRP2 knock-out mutant which were grown on medium supplemented withdifferent concentrations (0, 0.1 and 0.5 μM) of ABA hormone for 7 days,it could be understood that the mutant showed the tolerant (FIG. 6a ).As a result of comparison on germination rate in the wild type, theAtAIRP2 knock-out mutant and the AtAIRP2-overexpressing transgenic plantwhich were grown on medium supplemented with different concentrations(0, 0.2 and 0.4 μM) of ABA hormone for 7 days, it could be understoodthat the germination rates in the AtAIRP2-overexpressing transgenicplants were decreased by ABA hormone and the germination rate in theAtAIRP2 knock-out mutant was increased by ABA hormone (FIG. 6b ).

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present disclosure. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the disclosure as set forthin the appended claims.

1-13. (canceled)
 14. A method for improving the tolerance of a plant toan abiotic stress, comprising: (a) introducing a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:2 into a cell of theplant; and (b) obtaining a transgenic plant exhibiting improvedtolerance to an abiotic stress from the cell of the plant.
 15. Themethod according to claim 14, wherein the nucleotide sequence comprisesthe nucleotide sequence as set forth in SEQ ID NO:1.
 16. The methodaccording to claim 14, wherein the abiotic stress is selected from thegroup consisting of a drought stress, a low-temperature stress and asalt stress.
 17. The method according to claim 16, wherein the abioticstress is a drought stress.
 18. The method according to claim 16,wherein the abiotic stress is a low-temperature stress.
 19. The methodaccording to claim 16, wherein the abiotic stress is a salt stress. 20.The method according to claim 14, wherein the nucleotide sequence iscontained in a recombinant plant expression vector; and the recombinantplant expression vector comprises (i) the nucleotide sequence; (ii) apromoter which is operatively linked to the nucleotide sequence of (i)and generates a RNA molecule in plant cells; and (iii) a poly A signalsequence inducing polyadenylation at the 3′-end of the RNA molecule. 21.The method according to claim 14, wherein the plant is selected from thegroup consisting of food crops such as rice plant, wheat, barley, corn,bean, potato, Indian bean, oat and Indian millet; vegetable crops suchas Arabidopsis species, Chinese cabbage, radish, red pepper, strawberry,tomato, watermelon, cucumber, cabbage, melon, pumpkin, welsh onion,onion and carrot; crops for special use such as ginseng, tobacco plant,cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut andrape; fruit trees such as apple tree, pear tree, jujube tree, peachtree, kiwi fruit tree, grape tree, citrus fruit tree, persimmon tree,plum tree, apricot tree and banana tree; flowering crops such as rose,gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and foddercrops such as ryegrass, red clover, orchardgrass, alfalfa, tallfescueand perennial ryograss.
 22. The method according to claim 21, whereinthe plant is vegetable crops such as Arabidopsis species, Chinesecabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber,cabbage, melon, pumpkin, welsh onion, onion and carrot.
 23. The methodaccording to claim 22, wherein the vegetable crop is Arabidopsisspecies.
 24. The method according to claim 23, wherein the Arabidopsisspecies is Arabidopsis thaliana.
 25. A plant cell exhibiting improvedtolerance to an abiotic stress, transformed with the method according toclaim
 14. 26. A plant exhibiting improved tolerance to an abioticstress, transformed with the method according to claim 14.